Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is a neglected human pathogen of clinical significance. In addition, arenaviruses pose a biodefense threat. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and associated with severe side effects. Our long-term goal is to understand the molecular bases of HF arenavirus pathogenesis and to develop better antiviral strategies to combat these pathogens. We propose that the interaction between the virus precursor glycoprotein (GPC) and the S1P cellular protease may contribute to the pathogenesis of HF arenaviruses, and that S1P represents an attractive antiviral target. Our hypotheses are based on the following findings: 1) S1P-mediated processing of arenavirus GPC is critical for production of infectious progeny and virus propagation, and 2) S1P plays a critical role in the activation of several transcription factors implicated in the regulation of processes required for normal cell physiology, and high expression levels of GP in arenavirus infected cells may interfere with the normal activity of S1P, thus leading to altered cell physiology. Our specific aims are: 1. Biochemical and functional characterization of the arenavirus GP-S1P interaction. Arenavirus GPC and S1P co-localize within cells, but GP expression does not alter the normal sub-cellular distribution of S1P. We hypothesize that expression of arenavirus GP in infected cells can interfere with the processing and activation of S1P's cellular substrates involved in lipid metabolism regulation and ER stress response, thus contributing to arenaviral pathogenesis. To test this hypothesis we will characterize the formation and stability of the GP-S1P complex, and assess a possible competition between GPC and cellular substrate proteins for binding to the S1P protease. Likewise, we will use cell-based assays to uncover GP-induced perturbations on the normal S1P's cellular functions that could contribute to arenavirus pathogenesis. 2. Identification of small molecule inhibitors of S1P-mediated processing of arenavirus GPC. We will screen combinatorial libraries for candidate inhibitors of S1P-mediated cleavage of arenavirus GPC using two distinct HTS assay formats: 1) In vitro assays based on intra-molecular quenched fluorigenic (IQF) peptides derived from the S1P recognition sites of LASV GP and 2) cell-based assays monitoring Golgi- associated S1P activity as a readout. Selected candidates with high inhibitory potency (low EC90) and low cell toxicity (high CC50) will be subjected to counter screen protocols to select candidates that specifically inhibit S1P-mediated processing of LASV and JUNV GPs while having minimal effect on the processing of S1P's cellular targets. 3. Determination of the anti-viral potential of selected candidate compounds. We will test the hypothesis that candidate compounds with therapeutic index 10 (TI= CC50/EC50) will exhibit bona fide antiviral activity. For this we will determine the ability of these selected compounds to inhibit virus multiplication in cultured cells by specifically targeting S1P-mediated processing of GPCs of the pathogenic arenaviruses Lassa and Junin viruses. Inhibitor-escape mutants pose a general problem in antiviral therapy and thereby we will assess whether the emergence of viral variants resistant to inhibitors of S1P-mediated cleavage of GPC pose a significant obstacle to our proposed antiviral strategy. PUBLIC HEALTH RELEVANCE: Several arenaviruses, chiefly Lassa fever virus, cause severe hemorrhagic fever (HF) disease in humans, and evidence indicates that the worldwide-distributed prototypic Arenavirus LCMV is a neglected human pathogen of clinical significance. In addition, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapies are limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Therefore, the development of better antiviral strategies to combat pathogenic arenaviruses is highly relevant to human health, a task that is further complicated by the existing limited knowledge about the mechanisms underlying arenavirus pathogenesis. Our proposal is designed to examine the contribution of the GP-S1P interaction to arenavirus pathogenesis, and to provide a comprehensive assessment of the feasibility of inhibiting the S1P-mediated processing of the virus GPC as a novel antiviral strategy to combat arenavirus infections.